Journal: Molecular Cancer Research

Article Title: Metabolic Regulator IAPP (Amylin) Is Required for BRAF and RAS Oncogene-Induced Senescence

doi: 10.1158/1541-7786.mcr-20-0879

Figure Lengend Snippet: Figure 1. A lentiviral-based shRNA screen identifies IAPP as a new modulator of OIS. A, Schematic overview of genetic screen workflow. BRER cells were transduced with library, drug selected for stable integration and treated for 21 days with 4-OHT. Genomic DNA extracted from emergent clones was used to identify each shRNA contained therein. See also Supplementary Figs. S1 and S2 for BRER characterization and screening results. B, Representative photos of shIAPP cells and control cells after 7 dayswithout (EtOH) or with (þ4-OHT) BRAF activation. C, Flow cytometric analysis of cell-cycle distribution for cells treated as in B. D, Proliferation curves for BRER cells expressing vector (pRS19, left), control shRNA (pLKO-1 shLuc, right), or IAPP-specific shRNAs in the continuous presence of 4-OHT or solvent (EtOH) as indicated. Data are representative of three experiments, with biological triplicates for each time point. P < 0.05, P < 0.01, P < 0.001 (Tukey’s multiple comparisons test). E, RT-qPCR analysis of IAPP mRNA in HFTE cells transduced with shRNA to IAPP or indicated controls. Error represents SEM. Significance was determined using t test.

Article Snippet: BRER cells were infected with the DECIPHER Lentiviral shRNA Library, Human Mod 1: Signaling Pathway Targets (Addgene, #28285).

Techniques: shRNA, Transduction, Clone Assay, Control, Activation Assay, Expressing, Plasmid Preparation, Solvent, Quantitative RT-PCR

Journal: Molecular Cancer Research

Article Title: Metabolic Regulator IAPP (Amylin) Is Required for BRAF and RAS Oncogene-Induced Senescence

doi: 10.1158/1541-7786.mcr-20-0879

Figure Lengend Snippet: Figure 2. Loss of IAPP expression permits bypass BRAF-induced senescence. A, Western blot analysis of cell cycle and proliferation markers following 4 days of BRAF activation in vector control or shIAPP expressing cells. See Supplementary Fig. S3 for independent shRNA knockdown data. B, SAHF formation in BRER cells with and without shIAPP after 4 days 4-OHT treatment. Representative of more than three experiments. C, SA-b-Gal expression in vector control and shIAPP expressing BRER cells following 10 days 4-OHT treatment. Represen- tative of three experiments. D, Quantification of C from three independent experiments with a minimum of 450 cells assessed for each condition. Error bars represent SEM. Significance was determined with Student t test.

Article Snippet: BRER cells were infected with the DECIPHER Lentiviral shRNA Library, Human Mod 1: Signaling Pathway Targets (Addgene, #28285).

Techniques: Expressing, Western Blot, Activation Assay, Plasmid Preparation, Control, shRNA, Knockdown

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: Role of mTOR as an essential kinase in SCLC

doi: 10.1016/j.jtho.2020.05.026

Figure Lengend Snippet: Overview of the essential kinase screening strategy. SCLC tumor specimens were injected subcutaneously into NSG mice to obtain primary PDX. PDX were harvested, infected with the shRNA kinome library and cultured in vitro, or reimplanted into NSG mice. DNA was extracted from shRNA infected cells at specified time points (in vitro) or tumor volumes (in vivo), shRNAs PCR amplified, and next generation sequencing performed to quantify the retained shRNAs. Lost, or depleted, shRNAs were considered as essential kinase candidates and validated for their essential function in vitro and in vivo.

Article Snippet: A short-hairpin RNA (shRNA) kinome library consisting of 3,113 shRNAs targeting known human kinases (670 kinases) (TRC1.5 lentivirus-based shRNA library, The RNAi Consortium, Broad Institute) was obtained from the University of Colorado Cancer Center Functional Genomics Shared Resource Core.

Techniques: Injection, Infection, shRNA, Cell Culture, In Vitro, In Vivo, Amplification, Next-Generation Sequencing

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: Role of mTOR as an essential kinase in SCLC

doi: 10.1016/j.jtho.2020.05.026

Figure Lengend Snippet: Unsupervised hierarchical clustering of essential kinase profiles in MSK-LX-40. I/II-experiment number, Day 1/7/14=day of harvest post-shRNA infection of PDX cultured in vitro, Tumor=shRNA infected tumor grown in vivo in NSG mice, 1–3/4–6/7–9=sample triplicate libraries

Article Snippet: A short-hairpin RNA (shRNA) kinome library consisting of 3,113 shRNAs targeting known human kinases (670 kinases) (TRC1.5 lentivirus-based shRNA library, The RNAi Consortium, Broad Institute) was obtained from the University of Colorado Cancer Center Functional Genomics Shared Resource Core.

Techniques: shRNA, Infection, Cell Culture, In Vitro, In Vivo

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: Role of mTOR as an essential kinase in SCLC

doi: 10.1016/j.jtho.2020.05.026

Figure Lengend Snippet: Unsupervised hierarchical clustering of essential kinases from all PDX. I= experiment number, Tumor I/II/III=shRNA infected tumors grown in vivo in NSG mice.

Article Snippet: A short-hairpin RNA (shRNA) kinome library consisting of 3,113 shRNAs targeting known human kinases (670 kinases) (TRC1.5 lentivirus-based shRNA library, The RNAi Consortium, Broad Institute) was obtained from the University of Colorado Cancer Center Functional Genomics Shared Resource Core.

Techniques: shRNA, Infection, In Vivo

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-targeting control for MISSION shRNA library silences SNRPD3 leading to cell death or permanent growth arrest

doi: 10.1016/j.omtn.2021.09.004

Figure Lengend Snippet: Expression of SHC016 in MC38CEA results in apoptosis and cell-cycle arrest at the G2/M phase The expression of non-targeting shRNA SHC002 or SHC016 was induced by doxycycline (dox; 100 ng/mL) for the last 4, 3, or 2 days of the 5-day culture. (A) Cell viability assessed by MTT assay. Absorbance values of the cells without induced transgene expression were taken as 100%. (B) Analysis of death via apoptosis assessed by annexin V/PI staining and flow cytometry analysis; alive cells, annexin V − /PI − ; apoptotic, annexin V + /PI − ; and late apoptotic and necrotic, annexin V + /PI + and annexin V − /PI + (the latter usually did not exceed 3% of the cells expressing either shRNA). (C) Activity of caspase-3 and -7 measured via chemiluminescence assay. Chemiluminescence values of the cells without induced transgene expression were taken as 100%. (D) Flow cytometry analysis of the cell cycle. (B−D) The cells treated for the last 2 days of the experiment with etoposide (eto; 2 μM) were used as a positive control. (E) Western blot (WB) analysis of p53 levels in MC38CEA and MEF cells incubated for 48 h with or without dox. Luminescent signals were collected for approximately 3 min (MC38CEA) or 10 s (MEF). Representative images and quantifications of WB signals of three independent experiments are shown. Whole WB and Ponceau S-stained membranes are given in Figure S4 . (F) qRT-PCR analysis of mRNA levels of p53 target genes in MC38CEA and MEF cells incubated for 1 or 2 days with or without dox. The relative levels of the transcripts in uninduced cells were taken as 1. (A−F) Data are shown as mean values (MVs) from 3 independent experiments. Error bars represent standard deviation (SD).

Article Snippet: Engineered shRNA libraries have facilitated the development of high-throughput methods using arrayed or pooled RNAi screens to identify proteins involved in different cellular processes or novel specific therapeutic targets., , , A popular lentiviral TRC shRNA library has been developed by the RNAi Consortium at the Broad Institute and marketed by Merck (Darmstadt, Germany; previously Sigma-Aldrich, St. Louis, MO, USA) under the trade name MISSION.

Techniques: Expressing, shRNA, MTT Assay, Staining, Flow Cytometry, Activity Assay, Chemiluminescence Immunoassay, Positive Control, Western Blot, Incubation, Quantitative RT-PCR, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-targeting control for MISSION shRNA library silences SNRPD3 leading to cell death or permanent growth arrest

doi: 10.1016/j.omtn.2021.09.004

Figure Lengend Snippet: Expression of SHC016 in human cell lines results in inhibition of cell proliferation and/or viability (A) Cell viability assessed by MTT assay. The expression of non-targeting shRNA SHC002 or SHC016 was induced by dox (100 ng/mL) for the last 5, 4, 3, or 2 days of the 6-day culture. The measurements taken each day (A549, U251) or on the 5th day of dox treatment (PC3, MCF7, HeLa) are shown. Full data for PC3, MCF7, and HeLa are available in Figure S6 . Absorbance values of the cells without induction of shRNA expression were taken as 100%. (B) DNA synthesis and cell viability assessed via double BrdU/eFluor 520 viability staining were performed 3 (A549) or 5 (U251, PC3, MCF7, HeLa) days after inducing SHC002 or SHC016 expression. BrdU was added to the medium for the last 6 h of culture. The difference of the time schedule was dictated by shorter time needed to manifest cytostatic/cytotoxic effects of SHC016 in A549 and substantially shorter doubling time of A549 than of other cell lines ; dead, all eFluor 520 + cells; BrdU + , eFluor 520 − /BrdU + cells; and BrdU − , eFluor 520 − /BrdU − cells. (A and B) Data are shown as MV from 3 independent experiments. (A) Error bars represent SD. (C) Analysis of clonal growth capacity of A549, U251, and HeLa cells by colony formation assay. Pictures of culture plates with stained clones were taken 7–10 days after plating the cells. Expression of SHC002 or SHC016 was induced with dox, 24 h after seeding the cells. The number of colonies in the corresponding, uninduced cultures was taken as 100%. Data are shown as MV ± SD from 3 (U251 and HeLa) or 4 (A549) independent experiments.

Article Snippet: Engineered shRNA libraries have facilitated the development of high-throughput methods using arrayed or pooled RNAi screens to identify proteins involved in different cellular processes or novel specific therapeutic targets., , , A popular lentiviral TRC shRNA library has been developed by the RNAi Consortium at the Broad Institute and marketed by Merck (Darmstadt, Germany; previously Sigma-Aldrich, St. Louis, MO, USA) under the trade name MISSION.

Techniques: Expressing, Inhibition, MTT Assay, shRNA, DNA Synthesis, Staining, Colony Assay, Clone Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Non-targeting control for MISSION shRNA library silences SNRPD3 leading to cell death or permanent growth arrest

doi: 10.1016/j.omtn.2021.09.004

Figure Lengend Snippet: SHC016 expression cassette is eliminated from the cell cultures (A) Comparison of the viability of MCF7 and HeLa cells 5 days after induction of either SHC002 or SHC016 shRNA expression in the absence or presence of selection antibiotic, puromycin (1 μg/mL), which was added for the last 72 h of culture. Absorbance values of the cells without induction of shRNA expression were taken as 100%. Data are shown as MV ± SD from 3 independent experiments. (B) Comparison of DNA synthesis and cell viability assessed via BrdU/eFluor 520 viability staining of HeLa cells expressing for 5 days either SHC002 or SHC016 in the absence or presence of selection antibiotic, puromycin. Dead, all eFluor 520 + cells; BrdU + , eFluor 520 − /BrdU + cells; and BrdU − , eFluor 520 − /BrdU − cells. (C) PCR analysis of the abundance of the elements of pLKO vectors in the long-term cultures of MC38CEA and U251 cells. The cells were transduced with the empty vector (SHC001) or SHC002 or SHC016. DNA was isolated after indicated times, and the sequence from puromycin-resistance gene (puro) and the sequence comprising the shRNA insertion site (pLKO) were amplified. Inverted images of DNA electrophoresis gels are shown. S, size standard; GeneRuler 100 bp DNA Ladder, Thermo Scientific. The sizes of amplified fragments are as follows: puro, 123 bp; pLKO empty, 136 bp; and pLKO coding for either non-targeting RNA, 170 bp.

Article Snippet: Engineered shRNA libraries have facilitated the development of high-throughput methods using arrayed or pooled RNAi screens to identify proteins involved in different cellular processes or novel specific therapeutic targets., , , A popular lentiviral TRC shRNA library has been developed by the RNAi Consortium at the Broad Institute and marketed by Merck (Darmstadt, Germany; previously Sigma-Aldrich, St. Louis, MO, USA) under the trade name MISSION.

Techniques: Expressing, Comparison, shRNA, Selection, DNA Synthesis, Staining, Transduction, Plasmid Preparation, Isolation, Sequencing, Amplification, Nucleic Acid Electrophoresis

Journal: Cancers

Article Title: Applications of Genome-Wide Screening and Systems Biology Approaches in Drug Repositioning

doi: 10.3390/cancers12092694

Figure Lengend Snippet: Various RNA interference (RNAi) constructs used for temporary or permanent gene silencing and a general workflow of small interfering RNA (siRNA) screening towards drug repositioning (adapted from ). ( a ) siRNA, endoribonuclease-prepared siRNA (esiRNA), and long double-strand RNA (dsRNA), as naked RNAs, can be introduced to the cell through many pathways, such as lipid-based transfection reagents or electroporation, or active uptake by target cells through receptor-mediated endocytosis. Inside the cytoplasm, dsRNA is cleaved by endonucleases. Then, the obtained siRNA, along with the directly introduced siRNAs and siRNAs coming from the nucleus of hard-to-transfect cells which were achieved from viral vectors carrying siRNA or short hairpin RNA (shRNA) expression cassettes, are loaded into the RNA-induced Silencing Complex (RISC) to facilitate gene expression knock down. ( b ) Drug targets and available compounds are determined by in silico pathway analyses and database mining after two-step in vitro screening. Prior to clinical analysis, the efficiency of these drug candidates can be validated by in vitro and in vivo approaches.

Article Snippet: More recently, Takai et al. carried out a genome-wide RNAi through the GeneNet h50K shRNA library, which is composed of ~200,000 lentiviral shRNAs and more than 47,000 transcript targets, in order to identify genes with a synthetic lethal interaction with epithelial cellular adhesion molecule (EpCAM) as a potential therapeutic target for the EpCAM + AFP + Hepatocellular carcinoma subtype [ ].

Techniques: Construct, Small Interfering RNA, esiRNA, Transfection, Electroporation, shRNA, Expressing, Gene Expression, Knockdown, In Silico, In Vitro, In Vivo

Journal: Genes & Development

Article Title: TRPS1 acts as a context-dependent regulator of mammary epithelial cell growth/differentiation and breast cancer development

doi: 10.1101/gad.331371.119

Figure Lengend Snippet: TRPS1 loss impairs cell proliferation and lactogenic differentiation, mediated by increased interferon signaling. ( A ) Cell proliferation of control and TRPS1 knockout HC11 clones, as quantified using IncuCyte imaging for 140 h. Data represent mean ± standard error of the mean (SEM). ( B ) RT-qPCR analysis of β-casein ( Csn2 ) expression levels upon stimulation with DIP mix (dexamethasone, insulin, and prolactin) in control and TRPS1 knockout HC11 clones. Data represent mean + SD, n = 2. One-way ANOVA: (***) P < 0.001. ( C ) Pathway enrichment analysis performed on RNA-seq of HC11 control and TRPS1 knockout cells, using the MSigDB hallmark gene set collection . FDR < 0.05 was considered significant (blue bars). ( D ) Heat map reflecting gene expression changes at the genes listed in the interferon α and interferon γ gene sets indicated in C between HC11 control and TRPS1 knockout cells. Key factors in these signaling pathways are indicated. ( E ) Western blot analysis of STAT1 and STAT2 expression levels in HC11 control and TRPS1 knockout cells transduced with a nontargeting shRNA or a pool of shRNAs targeting Stat1 . β-actin was used as a loading control. ( F ) RT-qPCR analysis of β-casein ( Csn2 ) expression levels upon stimulation with DIP mix in control and TRPS1 knockout HC11 clones transduced with a nontargeting shRNA or a pool of shRNAs targeting Stat1 . Data represent mean + SD, n = 3. One-way ANOVA: (***) P < 0.001.

Article Snippet: Broad Institute's Mission TRC-1 mouse library lentiviral shRNAs were used targeting Trps1 or Stat1 ( Supplemental Table S2B ).

Techniques: Control, Knock-Out, Clone Assay, Imaging, Quantitative RT-PCR, Expressing, RNA Sequencing, Gene Expression, Protein-Protein interactions, Western Blot, Transduction, shRNA